Dramatic changes in systemic and renal hemodynamics occur during pregnancy. There is a marked decrease in systemic vascular resistance and reciprocal increases in cardiac output and global arterial compliance, accompanied by a modest decline in mean arterial pressure. The renal circulation participates in this maternal vasodilatory response, and consequently, renal plasma flow and glomerular filtration rate rise by 80 and 50%, respectively. Although the mechanisms underlying these maternal adaptations to pregnancy are not fully understood, there is increasing evidence that the ovarian peptide hormone relaxin plays a key role (reviewed in Conrad, K. P, 2011 “Emerging role of relaxin in the maternal adaptations to normal pregnancy: implications for preeclampsia” Semin Nephrol. 31(1):15-32).
Originally isolated from the ovary by Hisaw and colleagues, relaxin was named for its ability to relax the pubis symphysis in some species (Hisaw, F, 1926 “Experimental relaxation of the public ligament of the guinea pig” Proc Exp Biol Med 23:661-663). In non-human primates, it was subsequently shown to cause morphological changes in endothelial cells of endometrial blood vessels consistent with vascular hypertrophy and hyperplasia, and enlargement of arterioles and capillaries (Hisaw, F. L., Hisaw, F. L., Jr., and Dawson, A. B, 1967 “Effects of relaxin on the endothelium of endometrial blood vessels in monkeys (Macaca mulatta)” Endocrinology 81:375-385).
Relaxin circulates at low levels in the luteal phase of the menstrual cycle, and in pregnancy peaks during the first trimester falling to intermediate levels thereafter (Sherwood, O., 1994, Relaxin. NY: Raven. 861-1009 pp.). Previously relaxin was shown to augment MCP-1-induced monocyte chemotaxis, but was not a monocyte chemoattractant by itself (Figueiredo, K. A., Mui, A. L., Nelson, C. C., and Cox, M. E. 2006. Relaxin Stimulates Leukocyte Adhesion and Migration through a Relaxin Receptor LGR7-dependent Mechanism. J. Biol. Chem. 281:3030-3039).
Humans have three relaxin genes designated relaxin-1, -2 and -3 (Sherwood, O. D, 2004, “Relaxin's physiological roles and other diverse actions” Endocr Rev 25:205-234). Rats and mice each have two relaxin genes designated relaxin-1 and -3. Human relaxin-2, as well as rat and mouse relaxin-1 gene products are true orthologs, insofar as they are secreted by the corpus luteum during pregnancy and circulate. Humans, rats and mice have a relaxin receptor, the LGR7 (leucine rich repeat-containing G protein coupled) receptor recently renamed relaxin/insulin-like family peptide 1 receptor, RXFP1. Human relaxin may also bind to the LGR8 receptor (RXFP2), albeit with reduced affinity (Sudo S et al. 2003 “H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the actodomain and the exoloop 2,” J Biol Chem., 278(10):7855-7862). Recently, two new receptors have been described for relaxin-3, GPCR135 and 142 (Chen, J., Kuei, C., Sutton, S. W., Bonaventure, P., Nepomuceno, D., Eriste, E., Sillard, R., Lovenberg, T. W., and Liu, C, 2005, “Pharmacological characterization of relaxin-3/INSL7 receptors GPCR135 and GPCR142 from different mammalian species” J Pharmacol Exp Ther 312:83-95), although GPCR142 is a pseudogene in rats.
Infusion of recombinant human relaxin-2 (rhRLX) in nonpregnant conscious female and male rats significantly decreases renal and systemic vascular resistances, and increases cardiac output, renal blood flow, glomerular filtration, and global arterial compliance, thus mimicking the circulatory changes of pregnancy (Conrad, K. P., 2011 “Emerging role of relaxin in the maternal adaptations to normal pregnancy: implications for preeclampsia,” Semin Nephrol., 31(1):15-32). Conversely, administration of relaxin-neutralizing antibodies or ovariectomy inhibits the circulatory changes during midterm pregnancy in conscious rats (Conrad, K. P., Semin Nephrol. supra)). In addition to reductions in arterial tone and/or arterial compositional or geometrical remodeling, another likely mechanism for the decrease in systemic vascular resistance (SVR) and increase in global arterial compliance (AC) observed during relaxin administration or in pregnancy is increased angiogenesis (Conrad, K. P., Debrah, D. O., Novak, J., Danielson, L. A., and Shroff, S. G., 2004, “Relaxin modifies systemic arterial resistance and compliance in conscious, nonpregnant rats” Endocrinology 145:3289-3296).
Bone marrow derived endothelial cells (BMDEC) integrate into the vascular wall either differentiating into endothelial cells (vasculogenesis) or serving a paracrine role in stimulating local angiogenesis or endothelial repair. The number of CD34+ BMDEC expressing vascular endothelial growth factor receptor (VEGFR)-2 that circulate in the bloodstream is an independent predictor of early subclinical atherosclerosis in healthy subjects (Fadini, G. P., Coracina, A., Baesso, I., Agostini, C., Tiengo, A., Avogaro, A., and Vigili de Kreutzenberg, S. 2006. Peripheral Blood CD34+ KDR+ Endothelial Progenitor Cells Are Determinants of Subclinical Atherosclerosis in a Middle-Aged General Population. Stroke 37:2277-2282; Chironi, G., Walch, L., Pernollet, M.-G., Gariepy, J., Levenson, J., Rendu, F., and Simon, A. Decreased number of circulating CD34+KDR+ cells in asymptomatic subjects with preclinical atherosclerosis. Atherosclerosis In Press, Corrected Proof), inversely proportional to the risk factors for atherosclerosis (Vasa, M., Fichtlscherer, S., Aicher, A., Adler, K., Urbich, C., Martin, H., Zeiher, A. M., and Dimmeler, S. 2001. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 89:E1-7), and predicts future cardiovascular events (Schmidt-Lucke, C., Rossig, L., Fichtlscherer, S., Vasa, M., Britten, M., Kamper, U., Dimmeler, S., and Zeiher, A. M. 2005. Reduced Number of Circulating Endothelial Progenitor Cells Predicts Future Cardiovascular Events: Proof of Concept for the Clinical Importance of Endogenous Vascular Repair. Circulation 111:2981-2987).
Several studies have indirectly addressed the role of circulating BMDEC in postnatal vasculogenesis (Crosby, J. R., Kaminski, W. E., Schatteman, G., Martin, P. J., Raines, E. W., Seifert, R. A., and Bowen-Pope, D. F. 2000. Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation. Circ Res 87:728-730).
The number of circulating BMDEC in patients with both type I (quantifying by CFU) and type II (CD34+, VEGFR2+ cells measured by flow cytometry) diabetes is significantly reduced as compared with healthy subjects (Loomans, C. J. M., de Koning, E. J. P., Staal, F. J. T., Rookmaaker, M. B., Verseyden, C., de Boer, H. C., Verhaar, M. C., Braam, B., Rabelink, T. J., and van Zonneveld, A.-J. 2004. Endothelial Progenitor Cell Dysfunction: A Novel Concept in the Pathogenesis of Vascular Complications of Type 1 Diabetes. Diabetes 53:195-199; Werner, N., Kosiol, S., Schiegl, T., Ahlers, P., Walenta, K., Link, A., Bohm, M., and Nickenig, G. 2005. Circulating Endothelial Progenitor Cells and Cardiovascular Outcomes. N Engl J Med 353:999-1007). In addition, in vitro protocols used to evaluate the functional characteristics of diabetic BMDEC have demonstrated defective adherence (Fadini, G., Sartore, S., Schiavon, M., Albiero, M., Baesso, I., Cabrelle, A., Agostini, C., and Avogaro, A. 2006. Diabetes impairs progenitor cell mobilisation after hindlimb ischaemia-reperfusion injury in rats. Diabetologia 49:3075-3084; Capla, J. M., Grogan, R. H., Callaghan, M. J., Galiano, R. D., Tepper, O. M., Ceradini, D. J., and Gurtner, G. C. 2007. Diabetes impairs endothelial progenitor cell-mediated blood vessel formation in response to hypoxia. Plast Reconstr Surg 119:59-70), decreased ability to form tubes (Loomans, C. J. M., de Koning, E. J. P., Staal, F. J. T., Rookmaaker, M. B., Verseyden, C., de Boer, H. C., Verhaar, M. C., Braam, B., Rabelink, T. J., and van Zonneveld, A.-J. 2004. Endothelial Progenitor Cell Dysfunction: A Novel Concept in the Pathogenesis of Vascular Complications of Type 1 Diabetes. Diabetes 53:195-199; Tepper, O. M., Galiano, R. D., Capla, J. M., Kalka, C., Gagne, P. J., Jacobowitz, G. R., Levine, J. P., and Gurtner, G. C. 2002. Human Endothelial Progenitor Cells From Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation Into Vascular Structures. Circulation 106:2781-2786), and reduction in proliferative capacity (Tepper, O. M., Galiano, R. D., Capla, J. M., Kalka, C., Gagne, P. J., Jacobowitz, G. R., Levine, J. P., and Gurtner, G. C. 2002. Human Endothelial Progenitor Cells From Type II Diabetics Exhibit Impaired Proliferation, Adhesion, and Incorporation Into Vascular Structures. Circulation 106:2781-2786).
Fetal endothelial progenitor cells contribute to maternal angiogenesis during pregnancy (Nguyen Huu, S., Oster, M., Uzan, S., Chareyre, F., Aractingi, S., and Khosrotehrani, K. 2007. Maternal neoangiogenesis during pregnancy partly derives from fetal endothelial progenitor cells. Proc Natl Acad Sci USA 104:1871-1876). It is also possible that BMDECs participate in uterine vascular remodeling during gestation. Robb et al. hypothesized that since BMDECs may have a homeostatic role in maintaining both the maternal systemic and uterine vasculature, BMDEC may be the link between cardiovascular risk factors and increased risk of pre-eclampsia (Robb, A. O., Mills, N. L., Newby, D. E., and Denison, F. C. 2007. Endothelial progenitor cells in pregnancy. Reproduction 133:1-9).
However, endothelial progenitor cells are not abundant in either circulating blood or the bone marrow. In fact, the low abundance of endothelial progenitor cells represents one of the critical issues to overcome in the clinical application of endothelial progenitor cells. (Kawamoto et al. (2007) Catheterization and Cardiovascular Interventions 70:477-484.) Increased endothelial progenitor cell levels in the clinic currently are achieved by transplantation, which involves isolating endothelial progenitor cells from a donor, expanding the endothelial progenitor cells ex vivo, and then transplanting the endothelial progenitor cells to the recipient. Endothelial progenitor cell transplantation is an invasive and expensive procedure, and the ex vivo manipulation of isolated endothelial progenitor cells poses various health risks, such as transmission of infectious agents, to both the patient and care provider.
Failed or delayed fracture healing is a major clinical problem, and is mainly due to poor vascularization. Healing of bone fractures normally occurs within 8-12 weeks in healthy humans. However, a significant proportion of bone fractures fail to properly heal, i.e., non-union. A rate-limiting step in the healing process is blood flow, which is impeded by concomitant disruption of the bone vasculature. In addition, blood flow to the bone may be impaired at baseline before injury in the elderly, diabetics and smokers. The immobility resulting from bone fractures especially in the elderly is frequently fatal due to subsequent venous stasis, thrombosis and pulmonary embolism.
Neoangiogenesis is essential to bone healing. When disrupted by the angiogenic inhibitor TNP-470 normal osteogenesis was prevented resulting in pathologic fibrous union [Fang T D et al. 2005. Angiogenesis is required for successful bone induction during distraction osteogenesis. J Bone Miner Res. 20:1114-24]. Recent evidence shows that by enhancing mobilization of bone marrow progenitor cells, bone healing is accelerated in mice [Wang X X et al., 2011, Progenitor cell mobilization enhances bone healing by means of improved neovascularization and osteogenesis. Plast Reconstr Surg. 128:395-405; Matsumoto T et al., 2008, Fracture induced mobilization and incorporation of bone marrow-derived endothelial progenitor cells for bone healing. J Cell Physiol. 215:234-42; and Matsumoto T et al., 2006, Therapeutic potential of vasculogenesis and osteogenesis promoted by peripheral blood CD34− positive cells for functional bone healing. Am J Pathol. 169:1440-57]. Further increase was observed following their mobilization from bone by AMD-3100, which was associated with accelerated bone healing [Wang X X et al., 2011, ibid.].
The administration of human CD34+ cells to nude mice with experimentally induced fractures demonstrated enhanced vascularization, perfusion and bone healing associated with human specific markers for both endothelial cells and osteoblasts at the fracture site [Matsumoto T et al., 2006, ibid.]. In chimeric mice stably transplanted with bone marrow harvested from donors expressing LacZ regulated by the Tie-2 promoter, the same investigators demonstrated β-galactosidase staining at the fracture site [Matsumoto T et al., 2008, ibid.]. Taken together, these studies suggest an important role for vasculogenesis and osteogenesis mediated by bone marrow derived progenitor cells.
There is a need for methods effective at increasing bone marrow derived cells, including BMDEC and/or bone marrow-derived angio-osteogenic progenitor cell levels in the blood that do not require costly or invasive isolation or transplantation procedures. The present invention meets this need by providing novel methods useful for increasing bone marrow derived cells, particularly BMDEC mobilization.